ABSTRACT
PURPOSE: To use DNA microarray to analyze the expression patterns of genes in the uninoculated eye following uniocular anterior chamber inoculation of HSV-1. METHODS: On Day 9 following inoculation of 2 x 10( 4) PFU of HSV-1 (KOS strain) or an equivalent volume of tissue culture medium into one anterior chamber of BALB/c mice, the uninoculated eyes were enucleated, pooled, and total RNA was isolated. cDNA was synthesized from the total RNA. The gene expression patterns were inferred based on the hybridization intensities of the probes on the cDNA array. The hybridization signals were globally normalized and filtered. The data were analyzed using hierarchical and gene tree clustering algorithms. Additional uninoculated eyes collected on Day 9 p.i. were stained for F4/80 and CD19. RESULTS: Compared with the uninoculated eye of control mice, 3800 genes were upregulated at least twofold in the contralateral eye of HSV-1-infected mice. Among the 10 most upregulated genes, T cell-specific protein, MHC II antigen A, and MHC II k region locus 2 were upregulated 179-, 164-, and 162-fold, respectively. Ten T-cell receptor-related genes, 61 cytokine and chemokine genes, and 16 MHC genes were upregulated. Furthermore, 11 immunoglobulin and B cell genes and 11 macrophage-related genes were also upregulated. F4/80+ and CD19+ cells were observed on Day 9 p.i. CONCLUSIONS: The DNA microarray results support the idea that T cells and immunomodulatory factors (cytokines, chemokines) are likely to be involved in HSV-1 retinitis. These results also suggest that B cells and/or macrophages play a role in the pathogenesis of HSV-1 retinitis.
Subject(s)
Anterior Chamber/virology , Eye Infections, Viral/genetics , Gene Expression Regulation/physiology , Herpes Simplex/genetics , Herpesvirus 1, Human/physiology , Retinal Necrosis Syndrome, Acute/genetics , Animals , Antigens, CD19/metabolism , Antigens, Differentiation/metabolism , B-Lymphocytes/immunology , Eye Infections, Viral/metabolism , Eye Infections, Viral/virology , Female , Gene Expression Profiling , Herpes Simplex/metabolism , Herpes Simplex/virology , Immunoenzyme Techniques , Mice , Mice, Inbred BALB C , Oligonucleotide Array Sequence Analysis , RNA/isolation & purification , Retinal Necrosis Syndrome, Acute/metabolism , Retinal Necrosis Syndrome, Acute/virology , T-Lymphocytes/immunology , Up-RegulationABSTRACT
PURPOSE: To determine if apoptosis occurs in the eyes and brain following uniocular anterior chamber (AC) inoculation of HSV-1 and if expression of Fas ligand (FasL) or Fas is altered by virus infection. METHODS: After inoculation of HSV-1 via the AC route, apoptotic cells in the eyes and brain were identified by TUNEL assay, and TUNEL results indicating DNA fragmentation in the brain were confirmed by DNA laddering assays. Expression of FasL and Fas in the eyes and brain was determined by immunohistochemistry and Western blotting and confirmed by RT-PCR, Southern blotting of RT-PCR products, and sequencing. RESULTS: After AC inoculation of HSV-1, HSV-1 positive cells and apoptotic cells were observed in the eyes and brain. Immunofluorescent staining revealed that although both FasL and Fas were expressed in the cornea, retinal pigment epithelium cells (RPE), and photoreceptor cells of normal and virus infected mice, Fas and FasL were expressed at different locations in these cells. On day 8 p.i., more FasL DNA expression was observed in HSV-infected mice compared with uninfected mice. CONCLUSIONS: HSV-1 infection following AC inoculation of virus induced apoptosis in the eyes and brain. Although FasL expression was upregulated by HSV-1 infection, these results suggest there are likely to be factors in addition to FasL and Fas, such as Bcl-2 and caspases, that are involved in apoptosis observed in the eyes and brain of HSV-1-infected mice.
